We have studied early steps in homologous recombination carried out by a human recombinase activity isolated from nuclear extracts of HeLa cells. These early steps involve the recognition by recombinases of DNA sequence homology residing on two DNA molecules and the subsequent pairing of these sequences resulting in joint molecule formation. As few as 13 bp of sequence homology is recognized by the human recombinase protein in a joint molecule assay. The recombinase can pair a linear duplex with a homologous single-strand DNA which is either linear or circular. The reaction leads to the formation of stable joint molecules in which the two DNA substrates are joined by a region of hydrogen bonding. We have demonstrated that sequence recognition by recombinases does not require melting of the duplex DNA. Thermal stability assays demonstrate that the joint molecules do not have an unpaired strand that can participate in branch migration. On the basis of these observations, we conclude that a triple-stranded DNA structure is an intermediate in recombination. Efforts are underway to further characterize this novel protein-DNA complex.